ES2532408T3 - Procedure for purifying colistin and purified colistin components - Google Patents

Abstract

Procedure for the purification of colistin by reverse phase chromatography, characterized in that it presents the steps of loading a column based on colistin in 20-30% acetic acid and ethanol and eluting with 10-15% ethanol.

Description

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DESCRIPTION

Procedure for purifying colistin and purified colistin components.

Field of the Invention

The present invention relates to a method for the purification of antibiotics.

Background

The increase in multiresistance in gram-negative bacteria, in particular in Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, is a critical problem. The limitation of therapeutic options has forced doctors and microbiologists who are dedicated to infectious diseases to reevaluate the clinical application of colistin (also called polymyxin E), a polymyxin antibiotic similar, but not identical, to polymyxin B. Colistin may have advantages over polymyxin B due to its broader therapeutic index.

Colistin was first isolated in 1947 from Bacillus polymyxa var. colistinus and consists of a mixture of polypeptides produced by fermentation. The main components are polymyxin E1, E2, E3 and E1-Ile (Figure 1).

Commercially, colistin is found as colistin sulfate, which is used orally for intestinal decontamination and topically as a powder for skin infections, and as sodium colistimethate, which is used parenterally and by inhalation. It has been determined that colistimethate sodium is less toxic and has less undesirable side effects than colistin, but it is also less potent. (See Critical Care 2006, 10: R27 (doi: 10.1186 / cc3995), by Falagas and Kasiokou).

Colistin sulfate is often formulated in ointments, otic suspensions and otic and ophthalmic solutions. Colistin sulfate is also given orally in the form of suspensions or tablets to treat intestinal infections or to eliminate the flora of the colon.

Sodium colistimethate is a semisynthetic prodrug of colistin that can be used to treat Pseudomonas aeruginosa infections in patients with cystic fibrosis, and has recently been used to treat infections with multidrug Acinetobacter. Sodium colistimethate has also been administered intrathecally and intraventricularly in meningitis / ventriculitis due to Acinetobacter baumannii and Pseudomonas aeruginosa. Sodium colistimethate readily hydrolyses, resulting in a variety of methanesulfonated derivatives in aqueous solution, and is very difficult to analyze.

Since colistin was introduced into clinical practice more than 50 years ago, its properties were never documented as thoroughly as it is done with modern drugs, for example with specific requirements regarding its pharmacology, toxicology, impurity content, etc. For this reason, commercially available colistin products contain, in addition to their main component, polymyxin E1, many related active and inactive substances / impurities, mainly derived from the fermentation process. A typical HPLC chromatogram of colistin is shown in Figure 2.

Usually, the main component of commercial colistin, polymyxin E1, constitutes approximately 60% of the dry product. Some of the related substances in colistin products have been characterized (Figure 1), but most impurities remain unknown. The minimum potency of colistin sulfate, as specified by the USP (United States Pharmacopeia) is "not less than 500 µg / mg", but the specific antimicrobial activity of each component is largely unknown.

Although the product has been used for more than 50 years, there is no standardized dosage of colistin and no detailed trials on its pharmacology or pharmacokinetics have been published. Therefore, the optimal dosage of colistin for most infections is unknown. Similarly, the "maximum" dose recommended for each preparation is different. Each country has different generic preparations of colistin and the recommended dose depends on the manufacturer.

Colistin sulfate and sodium colistimethate can be administered intravenously and as aerosols, but the dosage is complex. Sodium colistimethate from some manufacturers is prescribed in international units, while the same product from other manufacturers is prescribed in milligrams of colistin base. This total absence of regulation or normalization of the doses makes intravenous dosing of colistin a nightmare for any doctor.

The main toxicities described in intravenous treatment are nephrotoxicity (kidney involvement) and neurotoxicity (nervous system involvement), but this may reflect the very high doses that were initially administered, much higher than those currently recommended by any manufacturer, and for 2 15

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which did not make any adjustment in case of kidney disease. The main toxicity described in aerosol treatment is bronchospasm.

In the absence of supporting data, it can be speculated that part of the toxicity of colistin can be attributed to the related substances and impurities present in current commercial products. In addition, when the colistimethate prodrug is synthesized using “impure” colistin as a starting material, each related substance and each impurity will form a base for various methanesulfonated derivatives, creating a very complex final product, in which the toxicological properties of each substance are unknown. individual.

From the above toxicological and pharmacological considerations, great advantages can be seen in the use of a "monocomponent" variety of colistin for medical purposes. Such monocomponent colistin will contain a very high proportion of the main polymyxin E1 component (> 90%, compared to the current 60%), and all major impurities will be identified and characterized.

A single component colistin offers several advantages:

1) Possibility to discern the toxicological contributions of the main component (PE1), related substances and impurities,

2) Product susceptible to new and more precise pharmacological and pharmacokinetic research,

3) Possibility of synthesizing well-defined derivatives, for example colistimethate, without giving rise to a multitude of poorly defined substances,

4) Possibility of developing better and more precise analytical methods for colistin and colistimethate, simplifying regulatory procedures,

5) Unambiguous dose recommendations based on clear pharmacological and toxicological data,

6) Update of colistin to make it a “modern” antibiotic, which is a great need.

De Crescenzo Henriksen et al (Lett Appl Microbiol 45, 491-496 (2007) describes the purification of polymyxin E from strains of P. amylolyticus in analytical grade by reverse phase HPLC purification steps, using gradients of mobile phase of isopropanol and methanol WO98 / 20836 discloses purification protocols of various components of colistin, including polymyxin E, using acetonitrile gradients in reverse phase HPLC.

A second objective, in the longer term, would be to subject purified PE1 to new efficacy studies in vitro and in vivo, as well as toxicological studies, in order to compare them with previous studies of the polymyxin group. Such randomized and controlled trials are urgently needed to clarify various issues related to the efficacy and safety of polymyxins (Crit Care Clin. April 2008; 24 (2): 377-91, by Micholopoulos and Falagas).

Characteristics of the invention.

The present invention relates to a process for purifying components of colistin, and particularly the main component, polymyxin E1.

The present invention relates to a process for producing a practically pure preparation of the main component of colistin, polymyxin E1 (90-98% purity), called monocomponent colistin.

Said process is a simple chromatographic method that uses solvents of low toxicity. The process comprises reverse phase chromatography (RP), which allows the purification of polymyxin E1 to a purity greater than 90%, followed by a hydrophobic interaction chromatography (HIC).

The procedure is characterized by

- loading a reverse phase column based on colistin in acetic acid and 20-30% ethanol,

- elution with 10-15% ethanol.

Surprisingly, the procedure can be used to efficiently separate polymyxin E1 from the other major components of colistin.

The work of identifying a suitable reverse phase material was carried out on a laboratory scale in

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10 x 250 mm steel columns.

Merck LiChrospher 60 RP-select B resin, 15 µm (C8) was found suitable for the purification of PE1 from the colistin base on a laboratory scale, and the laboratory method was transferred from the scale of one gram to that of fifty grams in a 50 x 850 mm steel column.

A procedure was developed in which the colistin base was dissolved in 24% ethanol (4 M) and 0.1 M acetic acid, and separated into polymyxin E fractions. The main component, PE1, was isolated with a Typical recovery of 60% and a relative chromatographic purity of 94-98%.

In European Pharmacopoeia (EP), the potency of a batch of colistin sulfate is defined as the content in% of the sum of the polymyxin factors PE1, PE2, PE3, PE1-Ile and PE1-7MOA, determined by HPLC in the state in which they are. The total power of these factors must constitute no less than 77.0%. In addition, the EP establishes maximum limits (NMT = no more than) for specific factors, such as: PE1-Ile (NMT 10%), PE1-7MOA (NMT 10%), PE3 (NMT 10%), and main impurity NMT 4 %.

Applying the purification method described, the final product has the following typical composition: PE1 (94-98%), PE2 (0-0.1%), PE3 (0.0%), PE1-Ile (0.2- 1.0%), PE1-7MOA (0.5-2.0%), and total impurities remaining 0.5-2.0%.

Brief description of the figures

Figure 1. Molecular structure of the main component of colistin sulfate, polymyxin E1 sulfate and related substances.

Figure 2. Colistin HPLC chromatogram, chemical reference substance of Ph. Eur.

Figure 3. The graphs illustrate the relative chromatographic purity as a function of the fraction number during the purification of the colistin base; f1-E1 and f2-E1 of the graph are collectively named f1 in the descriptive part of the application; eE1 is equivalent to e1 / e2, eeE1 is equivalent to e3, while eee ... is the sum of the unknown minor impurities.

Figure 4. The graphs illustrate the peak areas of the various components as a function of the fraction number during the purification of the colistin base; f1-E1 and f2-E1 of the graph are collectively named f1 in the descriptive part of the application; eE1 is equivalent to e1 / e2, eeE1 is equivalent to e3, while eee ... is the sum of the unknown minor impurities.

Figure 5. HPLC chromatogram of single component colistin (PE1) with major impurities.

Detailed description of the invention

The term "colistin" is intended to comprise any mixture of antibiotic peptide components in which the main component is polymyxin E1 or its salts. The term "polymyxin E1" is intended to comprise the compound previously designated colistin A, as well as the compound designated 7722-44-3 in Chemical Abstracts,

as well as the cyclic peptide (10-4) N2- (6-methyl-1-oxooctyl) -L-2,4-diaminobutanoyl-L-threonyl-L-2,4-diaminobutanoyl-L-2,4-diaminobutanoyl-L- 2,4-diaminobutanoyl-D-leucil-L-leucil-L-2,4-diaminobutanoyl-L-2,4-diaminobutanoyl-L-threonine,

as well as colistin IV. The term "colistin base" is intended to comprise any colistin comprising 30-70% E1 polymyxin. The term "colistin sulfate" is intended to comprise any salt of colistin sulfate. The term "colistimethate" is intended to comprise any methanesulfonated derivative of colistin. A "composition" is any mixture comprising more than two different compounds, for example, a mixture

of two active pharmaceutical principles, or a mixture of an active pharmaceutical principle and one or more excipients

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Pharmacists

The terms "component" or "components", used in the present application, refer to a specific compound of a composition. Accordingly, "minor components" are compounds that are found in relatively small amounts in a composition.

A "pharmaceutical composition" is any composition suitable for use in vivo. Such compositions can be administered cutaneously, subcutaneously, intravenously, parenterally, orally, etc.

"Separation" refers to any method in which a desired compound is separated from another compound (in analytical or preparative grade).

"Purification" is any method of separation whereby the concentration of a desired compound is increased.

"Chromatography" is any purification technique that comprises a stationary phase and a mobile phase.

A "stationary phase" is any surface comprising ligands capable of retaining compounds.

The "ligands" are residues of the stationary phase in which binding with the compounds occurs.

20 A "mobile phase" is any fluid, solvent, liquid or mixture that can pass through or along the stationary phase in a certain direction.

"Reverse phase chromatography" is any chromatography in which the more polar or charged components elute before the less polar ones.

"Hydrophobic interaction chromatography" is any chromatography based on the interaction between the apolar ligands of the stationary phase and the apolar compounds or the apolar part of the compounds.

"High concentration of ethanol" refers to a concentration of ethanol greater than or equal to 20% by volume, usually 20% -30%.

"Low ethanol concentration" refers to an ethanol concentration of less than 20% by volume, usually 10% -15%. 35 "% v / v" refers to volume percentage.

Commercial colistin base is a mixture of many closely related fatty acid and decapeptide amides, including polymyxin E1, polymyxin E2, polymyxin E3 and polymyxin E1-isoleucine (Figure 1).

40 The claim to isolate the main polymyxin E1 (PE1) component from the colistin base led to an investigation into whether a reverse phase HPLC (RP) method might or may not be suitable for the isolation of PE1 and obtaining the same with a high relative chromatographic purity (> 90%).

In the literature, only some methods of inverse phase separation are found, and the main organic solvents used are acetonitrile and methanol. These solvents are toxic and should be avoided in pilot and large-scale plant production. However, a microgram scale test of C18 column HPLC with colistin sulfate using a gradient of 0% to 60% ethanol indicated, surprisingly, that it might be possible to use the relatively non-toxic solvent ethanol for a purification method of polymyxin E1 useful

50 on an industrial scale.

The starting material, colistin base, is produced by fermentation and purification, and is an intermediate product in the production of colistin sulfate as a bulk substance. The starting material contains approximately 60% PE1, varying by a small percentage between batches. The original objective was to achieve purity

Relative chromatographic polymyxin sulfate E1> 90%. With this objective, a series of column materials were evaluated with elution systems based on ethanol.

Polymyxin E1 (Figure 1) has an ionic detergent character with a polar decapeptide part and an apolar fatty acid part. The molecule consists of a cyclic heptapeptide bound to a linear tripeptide acylated with 6-methyl octane.

60 Since the molecule contains 6 residues of 1,4-diaminobutyric acid (DAB), one of which participates in three peptide bonds, there are 5 primary amino groups in equilibrium with their corresponding ammonium groups (NH4 +), which constitute the strongly polar part of the molecule.

It was anticipated that the molecular interaction between the reverse phase resin and the molecule would take place in the rest of the fatty acid and the apolar regions of the peptide part.

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To monitor the HPLC sample groups and fractions, an analytical HPLC method was developed based on a standard method. The improved method revealed a small amount of related components (f1, e1, e2) below the main PE1 peak, which were invisible with the conventional HPLC method.

Experimental part

Preparative HPLC

10 x 250 column: For the evaluation of column materials on a milligram scale, a 10 x 250 mm steel column was used. The column was filled with the various resins under test suspended in 96% ethanol.

50 x 830 column: For preparative purification on a gram scale, a 50 x 850 mm steel column was used, as described below. The selected column material, approximately 1 kg of Merck LiChrosphere 60 RP select B (15 µm), was suspended in 96% ethanol and the column was filled. The upper flange was fixed and the piston was pushed up to 50 bar until all excess ethanol was removed. The column was evaluated by applying 1 ml of a 0.1 mg / ml potassium iodide solution, and the absorption was measured at 227 nm, AUFS = 0.05 and flow rate of 55.5 ml / min The recorded peak It was a narrow Gaussian curve with satisfactory symmetry.

The preparative HPLC system comprised:

Columns:

10 x 250 mm stainless steel Merck column and 50 x 850 mm stainless steel column with dynamic axial compression manufactured by Dan Process A / S

Pumps:

Waters Delta Prep 4000 with a flow range of 0.5-150 ml / min, with 4 different solvent inlets and with a mixing valve in the low pressure part

Detector:

Waters 486 Tunable Absorbance Detector

Integrator: Merck-Hitachi D-2000 Chromato-Integrator Fraction Collector: Waters Fraction Collector The absorbance of the eluent was detected at 230 nm, where there is a cut-off point for acetic acid. To minors

wavelengths, the eluent showed too much interference. The milligram scale fractions were collected in 25 ml test tubes, while the gram scale fractions were collected in 250 ml blue stopper bottles.

For the regeneration of the 10 x 250 mm and 50 x 850 mm columns after each HPLC cycle, a mixture containing 24% ethanol and 50% 1,2-propylene glycol in 0.1 M CH3COOH was used. Occasionally a high back pressure was observed with the 50 x 850 mm column, in which case it was repackaged or washed with 96% ethanol until the pressure was normal.

Analytical HPLC: A 4.6 x 150 Novapak column, 4 µm, C18, was used with acetonitrile as the mobile phase. The concentration of the

CH3CN solution was increased from 21% (after 10 minutes of isocratic cycle) to 30% over a time interval of 5 min. The columns were not thermostatted, but were operated at room temperature (23 ± 2 ° C). The analytical HPLC system comprised: Columns: Waters Novapak 4.6 x 150, 4µ C18 and equivalent column of 4.6 x 250 Pumps: 2 x Waters 510 with Waters Pump Control Module Detector: Waters 490 E Programmable Multi-wavelength Collector Detector of fractions: Waters 717 Plus Autosampler The system was controlled by Waters Millenium software. General description of the resins evaluated:

1) Merck No. 9303 LiChroprep RP-18, 25-40 µm, lot: L 275703 614. 6

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2) Merck No. 9324 LiChroprep RP-8, 25-40 µm, lot: L 240124 541.

3) Merck No. 11023 LiChrospher 60 RP-select B, 15 µm, (C8), lot: L 139923 633.

4) Merck No. 150385 Hibar Fertigsäule RT LiChrospher, RP-18, 15 µm, Cat. 50014.

5) Eka Nobel Kromasil-100 A, C8, 16 µm, lot: DT0026.

6) ToosoHaas Amberchrom CG 71 S, 35 µm, lot No. 23770319.

7) ToosoHaas No. 22227 Toyopearl MD-P Ether, 35 µm, weakly hydrophobic.

8) ToosoHaas No. 22225 Toyopearl MD-P Butyl, 35 µm, strongly hydrophobic. Chemicals for 10 x 250 column tests:

Colistin sulfate, lot: A4660314, Axellia ApS, Copenhagen, Denmark

Colistin base, lot: A1551701, Axellia ApS, Copenhagen, Denmark

Ethanol, 96%, “Danisco Distillers”, Danisco A / S

Methanol, Merck LiChrosolv No. 6018

Dimethylsulfoxide, Merck Uvasol No. 2950

Pure 1,2-propanediol, Merck No. 7478

2-propanol, LiChrosolv gradient grade, Merck No. 1040

Acetic acid, 100% G.R. Merck No. 63

1-methylpyrrolidone- (2) z.s., Merck No. 806072

Triethylamine, Picrce No. 25108

Tetrahydrofuran, Fluka No. 87367

Ammonium acetate p.a., Merck No. 1116

Ammonium Sulfate p.a., Merck No. 1217

Sulfuric acid 98% p.a.

Sodium hydroxide granules, GR, Merck # 6498

Milli-Q water, Lab purification, R&D, CDF, Axellia ApS, Copenhagen, Denmark

Millipore, membrane filter type HV, 0.45 µm. Chemicals for 50 x 825 column tests:

96% ethanol and 99.9% of Danisco Distillers, Danisco A / S

Acetic acid, 100% G.R. Merck No. 63

Sodium hydroxide granules, GR, Merck # 6498

NaOH 27%, dept. production

Potassium hydroxide, USP XIX, Ferak Berlin No. 20907 10 x 250 column tests: The following chromatographic resins were unsuitable for the separation task due to a joint

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strong with the resin, a pronounced phenomenon of the appearance of tails in the peaks, a low chromatographic purity or a low recovery: ToosoHaas Amberchrom CG 71 S; ToosoHaas Toyopearl MD-P Ether, 35 µm, ToosoHaas Toyopearl MD-P Butyl, 35 µm; Merck LiChroprep RP-8, 25-40 µm; Merck Hibar Fertigsäule RT LiChrospher, RP-18, 15 µm; Eka Nobel Kromasil-100 A, C8, 16 µm.

In the first 10 x 250 column tests with Lichroprep C18, 25-40 µm, with a gradient of 0% to 60% ethanol, for 60 min, at pH ~ 3.5 (50 mM HAc), with application of 11 mg of colistin sulfate, a relative chromatographic purity (RCP) of approximately 90% was obtained with good yields. However, when trying to reduce the concentration of ethanol by applying a gradient of 10-25% ethanol for 45 min and all other things being equal, no separation or any pronounced phenomenon of tails appeared on the peaks.

Similar tests with 5 mM NH4HSO4 at pH = 2.5 resulted in complete binding of the compounds to the column. This fact and similar experiments clearly indicated that colistin base should be used instead of colistin sulfate for the purification of PE1 under the selected conditions.

Surprisingly, if 1,2-propylene glycol (1,2-PG) was added as a substitute for part of ethanol, for example at a concentration of 24% ethanol and 20% 1,2-PG, and the amount of acid Acetic (pH = 3) was increased to approximately 1%, a product of PE1 was obtained with a CPR of approximately 90% with a yield of 70%.

From these initial experiments, it was concluded that acetic acid had a positive influence on the balance of compounds between the resin (LiChroprep C18) and the eluent, and that it was better to use colistin base dissolved in dilute acetic acid instead of sulfate Colistin However, the use of a mixture of 1,2-propylene glycol / ethanol resulted in a sharp drop in pressure with this column material, so that this resin and solvent mixture in particular were considered inadequate to transfer the tests to a greater scale.

It became clear that not only the rest of the fatty acid and the apolar amino acids participated in the column binding, but also the amino and ammonium groups.

Previous experiments yielded some surprising results, for example, that a) the ethanol solvent, respectful of the environment, health and safety (EHS), was useful as an eluent for the separation by reverse phase HPLC (RP) of the colistin, and that b) the separation should be made with colistin base in the presence of acetic acid instead of using colistin sulfate for further purification. Although LiChroprep C18 resin showed promising separation properties, it was not suitable for use on a larger scale.

The list of chromatographic media available on the market is very broad, but through careful screening and selection we identify a suitable Merck candidate, specifically the LiChrospher 60 RP-select B 15 µm column (C8).

The use of linear ethanol gradients, from 5% to 24%, in 0.1 M acetic acid gave promising separations with little appearance of tails in the peaks. Gradients of 5% to 15% ethanol in 0.1 M acetic acid (pH = 3) worked well, but with a CPR of only 85-90% PE1 and with low yields.

A breakthrough came when a gradient of inverted ethanol was tested (ie, application at high concentration of ethanol and elution at low concentration). The first tests with the following parameters gave a sample group with 90-95% PE1 and a yield of approximately 70%:

Balanced:

5% ethanol in 0.1 M acetic acid

Application:

110 mg of colistin base dissolved in 4 ml of 30% ethanol in 0.1 M acetic acid

Eluent:

15% ethanol in 0.1 M acetic acid at a temperature of 60 ° C

Temperature:

40 ° C

Eluent flow rate:

2.22 ml / min

A higher temperature was selected in order to more quickly reach the balance of the colistin components between the solid phase and the eluent, but the experiments showed that said higher temperature did not have a noticeable influence on the results. Consequently, the temperatures of the column furnace and the eluent phase were reduced from 40 ° / 60 ° to 35 ° / 50 ° without any significant change in the purity and performance of the sample group. Finally, a gradient of 5% to 15% ethanol in 0.1 M acetic acid at 30 ° / 40 ° was passed with good results, confirming that the inverted gradient was really responsible for the large positive change in chromatographic purity relative, performance and asymmetry profile of

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the peaks

It should be noted that during the development tasks a modified analytical HPLC method was implemented and that said new method revealed some related substances, f1 and e1 / e2, previously masked by the main peak 5 of E1.

The related substance f1 is eluted at the front of the PE1 peak, while substances e1 / e2 are eluted as a double peak with two more or less unfolded maxima in the tail of the PE1 peak. These two substances are particularly difficult to separate from PE1 and constitute a challenge in terms of purification in

10 the future optimization of the preparative HPLC method. See, in particular, Figure 3 and Figure 4, where the separation of PE1 and related substances is illustrated by diagrams showing the distribution of the components according to the HPLC fraction.

In Figure 3, the relative chromatographic purity of polymyxin E1 is compared with the related substances in

15 function of the fraction number. The most difficult related substances to separate from PE1 are e1 / e2, while E1-isoleucine, f1 and e3 (eluted after e1 / e2) are easier to remove. Figure 4 shows the results from the performance perspective, where the integration of the component peaks is represented as a function of the fraction numbers. The main sample group typically comprises fractions 7 to 12, but purity can be increased by reducing the selection of fractions.

20 Examples - Procedures for the purification of polymyxin E1 from the base of colistin

Example 1 -purification by reverse phase HPLC; mini-preparation scale

Equipment: Steel column 10 x 250 mm (ø = 10 mm) Waters Delta Prep 4000 Prep. Chrom. System Waters 4000 System Controller Waters 486 Tunable Absorbance Detector Waters Fraction Collector Merck Hitachi D-2000 Chromato-Integrator

Resin: LiChrospher 60 RP-select B Merck No. 11023.

Flow rate: 2.22 ml / min (increased to the scale of 300 x 700 mm ~ 2 l / min)

λ: 230 nm

AUFS: 2 (detector sensitivity ~ lowest level).

Buffer A: 12% ethanol (96%) in 0.10 M CH3COOH in Milli-Q water

Application sol: Colistin base, 600 mg ~ 0.5 mmol, dissolved in 15 ml of 24% ethanol (from 96% ethanol) in 0.10 M CH3COOH by adding 5 equivalents of CH3COOH acid ~ 2.5 mmol ~ 0.15 ml of 100% CH3COOH (17 M); adjusted with 2M NaOH at pH = 7.5; the solution is filtered by membrane (0.45 µm)

25 1) Column balancing: Balanced with buffer A.

2) Application: Colistin acetate solution (40 mg / ml) is applied at 2.22 ml / min, followed by 1 ml of buffer A through the feed tube. 30 3) Elution: The column is eluted immediately with buffer A and the effluent is collected in fractions of 1 ml ~ 2.22 ml.

4) Washing the column: 5 ml of 96% ethanol are applied, followed by buffer A until absorption 35 decreases to zero level.

5) HPLC analysis: The samples are diluted until the area of E1 reaches the value 20-30 x 106 AU; a high colistin standard usually reaches the value 20 x 106 AU; Samples are analyzed using Colistin NovaS (30 min cycle).

Example 2 -purification by reverse phase HPLC; pilot scale:

Equipment: 50 x 830 mm steel column (ø = 50 mm), “Dan-process” Waters Delta Prep 4000 Prep. Chrom. System Waters 4000 System Controller

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Waters 486 Tunable Absorbance Detector Waters Fraction Collector Merck Hitachi D-2000 Chromato-Integrator

Resin: LiChrospher 60 RP-select B, Merck No: 11023, 1 Kg

Flow rate: 65 ml / min (Increased to the scale of 300 x 700 mm ~ 2 l / min)

λ: 230 nm

AUFS: 2 (detector sensitivity ~ lowest level)

Buffer A: 12% ethanol (96%) in 0.10 M CH3COOH in Milli-Q water

Application sol: Colistin base, 50 g ~ 42 mmol (in the state it is in) is dissolved in 1,000 ml of 24% ethanol (96%) by adding 5.83 equivalents of CH3COOH acid ~ 245 mmol ~ 14.5 ml of 100% CH3COOH (17 M); the pH is adjusted with 2M NaOH at pH = 7.5; the solution is filtered by membrane (0.45 µm) using celite as a filter aid

Ionic strength: 4-6 mS / cm.

1) Balanced column: Balanced with buffer A.

2) Application: Colistin acetate solution (48 mg / ml) is applied at 65 ml / min for 14 min, followed by 5 buffer A for 0.1 min ~ 43.5 g applied

3) Elution: The column is eluted with buffer A until absorption is reduced to asymptote 0.010 V ± 10 mV and the effluent is collected in 18 fractions of 3 min ~ 585 ml.

10 4) Column washing: 200 ml (4 min at 50 ml / min) of a mixture of 24% ethanol and 1.2 50% propanediol are applied, followed by buffer A until absorption decreases to zero level .

5) HPLC analysis: The samples are diluted until the area of E1 reaches the value 20-30 x 106 AU (5x); a high colistin standard usually reaches the value 20 x 106 AU; Samples are analyzed using 15 Colistin NovaS (30 min cycle).

Example 3 - Total purification and final handling; big scale

Monocomponent colistin sulfate (polymyxin E1) is a fermentation product, which implies that its

First appearance occurs in a fermented broth. Polymyxin E1 is recovered and purified from the broth that contains a wide variety of impurities and only a few grams of polymyxin E1 and related substances per liter.

Recovery from the fermentation broth comprises the precipitation of colistin (polymyxin E1) and

25 related substances, and a primary separation by centrifugation. Secondary purification consists of reverse phase chromatography and precipitation, which give rise to the purest product with respect to polymyxin E1. The related substances and impurities present in the monocomponent colistin sulfate product are mainly co-fermented substances.

30 During the fermentation of colistin, a complex mixture of structurally related components is generated, with a characteristic ratio of each strain of Bacillus polymyxa. Examples of this are:

-Changes in the fatty acid chain that generate, for example, polymyxin E1 (with 6-methyl-octanoic acid) or polymyxin E2 (with 7-methyl-octanoic acid), 35

-Amino acid substitution in the molecule; for example, if the colistin D-leucine is replaced by Dphenylalanine, the polymyxin B related antibiotic is generated. If the polymyxin E1 L-leucine is replaced by isoleucine, E1-isoleucine polymyxin is generated.

40 Purification by preparative HPLC

The following buffers are prepared:

Buffer I: 1.6-2 M ethanol and 0.1-0.15 M acetic acid. The buffer is filtered through a 0.45 µm filter immediately before use.

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Buffer II: 1,2-propanediol approximately 6.8 M, ethanol approximately 4 M and acetic acid approximately 0.1 M. The buffer is filtered through a 0.45 µm filter immediately before use.

The HPLC column (diameter of 30 cm), with a volume of approximately 48 liters, is equilibrated with approximately 40 liters of buffer I. The flow rate is 0.8-1.2 l / min (the same flow rate is used for all stages of HPLC). The column is regenerated with approximately 8 liters of buffer II, followed by approximately 50 liters of buffer I or until the UV signal returns to the starting value.

A batch of vacuum dried colistin base, 1000-1,500 g, is weighed and suspended in 20-30 liters of 4M ethanol. The suspension is dissolved by the addition of 0.3M acetic acid while stirring for 30 min. . The pH is adjusted to approximately 7.5 with sodium hydroxide and the solution is filtered through a membrane filter, 0.45 µm. The colistin acetate solution is passed through the HPLC column and binds to the resin. The HPLC column is eluted with approximately 160 liters of buffer I. The effluent is collected in fractions. Fractions that meet a predetermined specification are collected and the others are discarded. A batch with a purity of at least 92% with respect to polymyxin E1 is grouped from the fractions. A concentration of polymyxin E1 and the removal of excess ethanol by reverse osmosis is carried out. The sample group is concentrated to approximately 50 g / l and then dialyzed with deionized water (approximately 8 volumes).

Purification by hydrophobic interaction chromatography (HIC)

The following buffers are prepared:

Calibration buffer: (NH4) 2SO4 approximately 200 mM; the pH is adjusted to approximately 7 with dilute ammonia;

Elution buffer: (NH4) 2SO4 approximately 200 mM; The pH is adjusted to approximately 3.7 with acetic acid.

The HIC column (diameter of 35 cm), with a volume of approximately 90 liters, is equilibrated with 250 liters of the calibration buffer, with a flow rate of approximately 1.7 l / min, followed by approximately 200 liters of elution buffer , with a flow rate of approximately 1.0 l / min.

The saline content in the colistin acetate solution from reverse osmosis is adjusted to approximately 200 mM by the addition of (NH4) 2SO4 and the pH is adjusted to approximately 7 with dilute ammonia. The concentration of colistin acetate solution is 15-20 g / l. The solution is passed through the HIC column, with a flow rate of 950-1.050 ml / min. Elution occurs with 10 times the bed volume. The collection of the effluent fractions is carried out automatically by PLC. Samples of each fraction are taken and analyzed. Fractions that meet the predetermined specifications are combined, obtaining a batch with a purity of 94-98% with respect to polymyxin E1. A concentration of polymyxin E1 and the removal of ammonium sulfate by ultrafiltration to a concentration of 10-20 g / l is carried out.

Precipitation of the single component colistin base

The solution is diluted to 10 g / l with deionized water and stirred until homogeneous. The pH is adjusted to 9.6-9.8 with sodium hydroxide and stirring is continued while the monocomponent colistin base precipitates. The precipitate is recovered by filtration in a filter press. When filtration is complete, the cake is washed with approximately 800 liters of deionized water, which is displaced by air. The single-component colistin cake is extracted from the filter press and stored in a freezer.

Conversion to monocomponent colistin sulfate

The monocomponent colistin base is suspended and dissolved in deionized water with stirring. The pH is adjusted to 5.0 with dilute sulfuric acid. The solution is filtered through a 0.45 µm filter.

Freeze drying, grinding and storage

The filtered solution is introduced into stainless steel freeze-drying trays and lyophilized for approximately 70 hours with a temperature profile controlled by PIC within the range -25ºC → + 45ºC. The dried product is extracted from the lyophilizer and ground to obtain the desired particle distribution.

Analytical HPLC methods

Method 1: HPLC for control during the process from fermentation to colistin base

Equipment: Waters automatic HPLC equipment, comprising:

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Pump: Model 6000 A / 510 Injector: WISP 712 A Detector: Model 441 Prefilter: Guard Pak, Resolve C18 (can be omitted) Column: Resolve C18, 10 x 8.5 µm or Nova-Pak, 4 µm Column mount: RCM 8 x 10

Method: Isocratic

Mobile phase: Buffer: Sodium sulfate decahydrate, 16.1 g (0.05 M), conc. Acetic acid, 0.56 ml (0.01 M), triethylamine, 20 ml (0.15 M), water up to 1,000 ml The pH is adjusted to 2.5 with phosphoric acid. Alternative buffer: 0.04 M sodium sulfate, 0.56 M methanesulfonic acid, 0.087 M triethylamine, pH at 3.0. Before use, acetonitrile, 170 g, is dissolved in the buffer to have 1,000 ml. The solution is purged with helium for 15 min.

Procedure of

test: Flow rate: 1.5 ml / min, alternative 1.0 ml / min Temperature: ambient. Injection volume: 20 µl, alternative 25 µl. Detection: 214 nm, 0.1 AUFS or 1.0 AUFS. Duration: 30 min. Standard: Authentic colistin sulfate standard, dissolved in water up to 1 mg / ml.

Standard: Colistin base, 1.0 mg / ml in 0.1 M hydrochloric acid.

Analytical samples: Dilute to contain 0.5-1 mg / ml. Diluent: 3% phosphoric acid, 40 ml, and acetonitrile, 60 ml. Centrifugation if turbidity is observed.

Method 2: HPLC for colistin base to final bulk product

Equipment: Waters automatic HPLC equipment, comprising:

Pump: Model 510 Injector: 717 Plus autosampler Detector: 490 E Prefilter: Guard Pak, Nova-Pak, C18 Column: Nova-Pak, C18, 60 Ǻ, 4 µm, 150 x 4.6 mm. Column oven: Jones Chromatography Model 7955

Reagents:

Anhydrous sodium sulfate for analysis Triethylamine, HPLC grade Methanesulfonic acid, ≥ 99% Water Milli-Q Buffer: Anhydrous sodium sulfate, 28.4 g (0.10 M), triethylamine 14.0 ml (0.05 M), 10.0 ml methanesulfonic acid (0.06 M), Milli-Q water up to 2,000 ml. The buffer is filtered under vacuum through a 0.45 µm filter. Eluent A: 50% buffer, 35% Milli-Q water, 15% acetonitrile. Adjusted to a pH of 2.0 with methanesulfonic acid. Eluent B: 50% buffer, 20% Milli-Q water, 30% acetonitrile. Adjusted to a pH of 2.0 with methanesulfonic acid.

Sample preparation:

Colistin acetate: 1-2 g / l in acetic acid 0.05 M. Stirring for 30 minutes and filtration through a 0.45 µm membrane filter

Patterns:

Two own standards are prepared, one with high activity (2,000 µg / mg) and one with low activity (400 µg / mg).

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Test procedure:

Flow rate: 0.8 ml / min Column temperature: 25ºC Detection: 214 nm Time constant: 1.0 s AUFS: 1.000 AU Auto zero: On WISP temperature: 25ºC. Injection: 20 µl / cycle

Gradient Program:

Gradient:

Weather Flow rate (ml / min) %TO % B %C Curve

one

0.00 0.8 67.0 33.0 0.0 0

2

15.00 0.8 67.0 33.0 0.0 6

3

25.00 0.8 30.0 70.0 0.0 6

4

30.00 0.8 30.0 70.0 0.0 6

5

31.00 0.8 67.0 33.0 0.0 6

6

40.00 0.8 67.0 33.0 0.0

6

Changes after 40 minutes are not part of the trial, they only take place after a series of analyzes

7

41.00 0.8 0.0 100.0 0.0 6

8

50.00 0.8 0.0 100.0 0.0 6

9

51.00 0.00 0.0 100.0 0.0 6

Eluent A is used as blank and is subtracted from patterns and samples.

Method 3: HPLC for single component colistin sulfate

Equipment: Waters automatic HPLC equipment, comprising:

Pump: Model 510 Injector: 717 Plus autosampler Detector: 490 E Column: Nova-Pak, C18, 60 Ǻ, 4 µm, 250 x4.6 mm Column oven: Jones Chromatography Model 7955

Reagents:

Water Milli-Q Acetonitrile, HPLC grade Methanesulfonic acid, ≥ 99% Triethylamine, HPLC grade Anhydrous sodium sulfate for analysis Buffer: Anhydrous sodium sulfate, 28.4 g (0.10 M), triethylamine 14.0 ml (0, 05 M), 10.0 ml methanesulfonic acid (0.06 M), Milli-Q water up to 2,000 ml. The buffer is filtered under vacuum through a 0.45 µm filter. Eluent A: 50% buffer, 35% Milli-Q water, 15% acetonitrile. Adjusted to a pH of 2.0 with methanesulfonic acid. Eluent B: 50% buffer, 20% Milli-Q water, 30% acetonitrile. Adjusted to a pH of 2.0 with methanesulfonic acid.

Sample preparation:

Monocomponent Colistin Sulfate: 2.8 mg / ml in Milli-Q water. Stirring for 15 minutes and filtration through a 0.45 µm filter.

Test procedure:

Flow rate: 1.0 ml / min Detection wavelength: 214 nm Column temperature: 25ºC Injection volume: 40 µl

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Autosampler temperature: 25ºC Gradient program:

Gradient:

Weather Flow rate (ml / min) %TO % B Curve

one

0.00 1.00 67.0 33.0 0

2

20.00 1.00 67.0 33.0 6

3

40.00 1.00 40.0 60.0 6

4

50.00 1.00 40.0 60.0 6

5

51.00 1.00 0 100.0 6

6

55.00 1.00 0 100.0

6

7

56.00 1.00 67.0 33.0 6

8

70.00 1.00 67.0 33.0 6

Changes after 70 minutes are not part of the trial, they only take place after a series of analyzes

9

71.00 1.00 0 100 6

10

81.00 1.00 0 100 6

eleven

82.00 0 0 100 6

5 References

1) Suzuki, T., Hayashi, K., Fujikawa, K. (1963), Studies on the Chemical Structure of Colistin: III. Enzymatic Hydrolysis of Colistin A, J Biochem 54: 412-418.

10 2) Elverdam, I., P. Larsen and E. Lund (1981), Isolation and characterization of three new polymyxins in polymyxin B and E by high-performance liquid chromatography. J. Chrom. (218) 653-661.

Claims (8)

1. Procedure for the purification of colistin by reverse phase chromatography, characterized in that it presents the stages of 5 Load a column based on colistin in acetic acid and 20-30% ethanol and elute with 10-15% ethanol. 2. The method according to claim 1, wherein the concentration of ethanol is from 20% to 24%. 3. Method according to claim 1, wherein the concentration of ethanol is 24%. 4. Process according to claim 1, wherein the concentration of ethanol is 12%.

5.

Process according to claim 1, wherein the concentration of acetic acid is 0.1 M.

6. Method according to claim 1, followed by an additional step of hydrophobic interaction chromatography.

7.

Method according to claim 1, wherein the pH of the mobile phase is 7 to 8. 20

8. The method according to claim 1, wherein the pH of the mobile phase is 7.5. 9. The method according to claim 1, wherein the high concentration of ethanol is 24%, the low concentration of ethanol is 12%, The concentration of acetic acid is 0.1 M, The pH of the mobile phase is 7.5, and the stationary phase is a C8 resin. fifteen